Use of alpha 1AR subtype-selective drugs in patients with acute myocardial infarction

ABSTRACT

The present invention relates to the use of α 1a AR-selective and/or α 1a /α 1d -selective antagonists in a method of preventing restenosis after myocardial infarction and re-perfusion. The invention further relates to a method of identifying agents suitable for us in such a method.

[0001] This application claims priority from Provisional Application No.60/169,294, filed Dec. 7, 1999, the entire content of which isincorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to the use of α_(1a)AR-selectiveand/or α_(1a)/α_(1d)-selective antagonists in a method of preventingrestenosis after myocardial infarction and re-perfusion. The inventionfurther relates to a method of identifying agents suitable for us insuch a method.

BACKGROUND

[0003] Alpha₁-adrenergic receptor (α₁AR) stimulation mediatessympathetic nervous system responses such as vascular smooth musclecontraction and myocardial hypertrophy. α₁AR-mediated vasoconstrictioncontributes to baseline (tonic) vessel tone, modulates systemic vascularresistance/venous capacitance, and is important in cardiovascularresponses to shock.¹ in addition, during “fight and flight” responses,elevated catecholamines result in constriction of “nonessential”vascular beds (e.g. splanchnic) while blood flow to vital organs (e.g.,brain, heart) remains uncompromised.^(2,3) cDNAs encoding three humanα₁AR subtypes (α_(1a), α_(1b) and α_(1d) ^(4,5)) were recently cloned,each expressed receptor pharmacologically characterized,⁴ and speciesheterogeneity in α₁AR subtype tissue distribution identified.^(6,7) Allthree α₁ARs couple predominantly via Gq to phospholipase C-b activation,resulting in formation of inositol trisphosphate (IP₃), calcium releasefrom intracellular stores, and ultimately to smooth muscle contraction.⁸

[0004] Although reasons for existence of three α₁AR subtypes remainelusive, recent findings suggest subtype and tissue specific regulationmay be important.^(9,10) While all α₁AR subtypes mediate smooth musclecontraction, hypertrophic pathways demonstrate subtype specificsignaling.¹¹ α₁AR agonist exposure to neonatal rat myocytes results inα_(1a)AR mRNA/protein upregulation (doubling) concurrent with α_(1b) andα_(1d) downregulation, correlating with induction of myocardialhypertrophy.¹² In contrast, insulin and insulin-like growth factor Iinduces α_(1d)AR expression in cultured rat vascular smooth musclecells.¹³ Hence agonist exposure, disease states, and drugs alter α₁ARsubtype expression.

[0005] The present invention results, at least in part, from studiesdesigned to determine the mechanisms underlying cardiovascular responsesto acute stress and chronic catecholamine exposure (e.g. aging). Humanvascular a₁AR subtype distribution and function were examined.Specifically, two hypotheses were tested: 1) human α₁AR subtypeexpression differs with vascular bed, and 2) age influences humanvascular α₁AR subtype expression. The results demonstrate human vascularα₁AR subtype distribution differs from animal models, varies with vesselbed, correlates with contraction in mammary artery, and is modulated byaging.

SUMMARY OF THE INVENTION

[0006] The present invention relates to the use of α_(1a)AR-selectiveand/or α_(1a)/α_(1d)-selective antagonists in a method of preventingrestenosis after myocardial infarction and re-perfusion. The inventionfurther relates to a method of identifying agents suitable for us insuch a method.

[0007] Objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A-1C—Representative saturation binding isotherms for humanmammary artery (FIG. 1A), aorta (FIG. 1B), saphenous vein (FIG. 1C).

[0009]FIG. 2—Representative RNase protection assay. Autoradiograph(24-hour exposure) demonstrating specific hybridization of a₁AR subtyperadiolabeled antisense riboprobes with total RNA isolated from tRNA(negative control), human aorta, vena cava, iliac artery, and renalartery.

[0010] FIGS. 3A and 3B—Phosphorimager counts from RNase protectionassays for each a₁AR subtype. a_(1a)AR mRNA expression is significantlyhigher overall in arteries (FIG. 3A) compared with a_(1b)AR and a_(1d)AR(**p<0.001), particularly splanchnic (SPL) versus central arteries (CEN)(*p<0.05) (FIG. 3B).

[0011] FIGS. 4A-4D—Phenylephrine-induced mammary artery contraction.Antagonists prazosin (nonselective) (FIG. 4A), 5-MU (a_(1a)-selective)(FIG. 4B), spiperone (relatively a_(1b)-selective) (FIG. 4C) demonstrateconcentration dependent shift in potency without reducing maximumresponse. BMY7378 (α_(1d)-selective) (FIG. 4D) does not produce asignificant shift. Two concentrations of antagonist are shown: ▪control; ▴ low (prazosin—10⁻⁹ mol/L; 5-MU/spiperone/BMY7378—10⁻⁸ mol/L); higher (prazosin—10⁻⁸ mol/L; 5-MU/spiperone/BMY7378—10⁻⁷mol/L).

[0012] FIGS. 5A-5D—a₁AR subtype expression in mammary artery from young(<55 years, n=6) (FIGS. 5A and 5C) versus older (>65 years, n=6) (FIGS.5B and 5D) patients. Competition analysis with 5-MU(a_(1a)>a_(1b)=a_(1d)) (FIGS. 5A and 5B) or WB4101(a_(1a)=a_(1d)>a_(1b)) (FIGS. 5C and 5D). See Table 4 for pKivalues.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention results, at least in part, from studiesdesigned to characterize a₁AR subtype distribution in humans acrossdifferent vascular beds. The results presented in the Example thatfollows demonstrate a₁AR subtype expression varies according to vesselbed. Specifically, a_(1a)AR mRNA/protein predominates in coronary,splanchnic, renal, and pulmonary arteries, whereas central arteries andveins express all 3 a₁ARs. With aging (<55 versus >65 years), a two-foldincrease in overall mammary artery (but not saphenous vein) a₁ARexpression occurs (a_(1b)>a_(1a)). Robust α_(1a) and α_(1b)-mediatedcontraction for all ages studied indicate these findings have functionalsignificance.

[0014] α₁AR-mediated smooth muscle contraction is important indetermining tonic and reflex changes in arterial and venous diameter.Instantaneous changes in vessel tone are responsible for maintenance ofblood pressure and venous return to the heart during stress (e.g.hypovolemia [hemorrhage], shock, and sepsis).¹ At rest, adult splanchnicvessels contain 30% total circulating blood volume;³ acutesympathetically-mediated constriction is a primary mechanism underlyingmaintenance of blood pressure during shock or hemorrhage. Robustness ofcompensatory mechanisms is illustrated by blood pressure stabilityuntil >20% blood volume is lost.¹⁸

[0015] Vascular α₁ARs have been studied in animals using a variety oftechniques (Table 5). After initial controversy, it has been generallyagreed a_(1d)ARs mediate vasoconstriction in rat aorta;^(15,19) incontrast, contraction in dog, rabbit, and mouse aorta occurs viaa_(1b)ARs.²⁰⁻²² a₁AR subtype-mediated contraction also differs alongmesenteric bed; a_(1d)AR mediates contraction in rat superior mesentericartery (proximal) whereas a_(1b)ARs function in distal mesentericarteries.¹⁹ Only a few studies in human vessels have been performed todate; these identify all three a₁AR subtype mRNAs in human mesentericartery,²³ a_(1b) and α_(1d) in human aorta,⁶ and a_(1a) in saphenousvein²⁴ and vena cava.⁶ a_(1b)-mediated contraction occurs in humansuperior vesicle and obturator arteries,²⁵ and a_(1a)-mediatedcontraction in human mesenteric artery.²⁶ The findings further indicatethat α_(1a)AR-mediated contraction accounts for generalized splanchnicvasoconstriction during stress in humans, although this hypothesis mustbe confirmed by further contraction studies. Other findings of clinicalrelevance include a₁ARs in renal, pulmonary, and coronary vasculature aspossible targets for treatment of renal insufficiency, pulmonaryhypertension, and angina. Since veins contain all three a₁AR subtypes,pharmacological isolation preload (venous return) and afterload(arterial vascular resistance) is possible. While the experimentsdescribed in the Examples that follow utilized “normal” vessels,examination of alterations of a₁AR subtype distribution by disease canbe made.

[0016] Sympathetically-mediated vascular responsiveness changes withage, although precise mechanisms underlying this observation remainunknown.⁸ While overall aortic a₁AR density remains unchanged with agein rat, subtype modulation occurs (increased a_(1a), decreasedα_(1b),unchanged α_(1d));²⁷ other studies suggest age decreases all a₁ARs inrat,²⁸ but increases in sheep.²⁹ Age-related changes are vesselspecific, with rat renal α_(1b)AR mRNA declining without change inmesenteric/pulmonary a₁ARs.²⁸ Furthermore, age increases functionala_(1d)ARs in resistance vessels compared with a_(1a)AR predominance inyoung rats.³⁰ In humans, age increases in-hospital mortality associatedwith major surgery;³¹ risks include vascular-associated conditions suchas gastrointestinal infarction and limb ischemia.^(2,32,33) The resultsreveal age-related increases in mammary artery a₁AR density (but notsaphenous vein), and a switch from a_(1a) predominance in younger adultsto a_(1b)>a_(1a) in older patients. Other arteries need to be tested todetermine whether age-induced arterial changes are global, or mammaryartery specific. In support of a global interpretation of the presentfindings, a recent clinical study demonstrates less blood pressureperturbation in elderly patients with tamsulosin(a_(1a)/a_(1d)-selective antagonist) compared with alfuzosin(non-selective),³⁴ indicating importance of a_(1b)ARs with aging inresistance vessels.

[0017] The result presented herein demonstrate human vascular α₁ARsubtype distribution differs from animal models, varies with vessel bed.correlates with contraction in mammary artery, and is modulated byaging. This information provides targets for therapeutic intervention ina clinical settings.

[0018] Certain aspects of the present invention are described in greaterdetail in the Example that follows.

EXAMPLES

[0019] Methods

[0020] Human Vessels

[0021] Vessels were obtained after approval from the Duke Universityinstitutional review board and individual agencies. Sources includeddiscarded tissues from surgery (0-60 minutes from isolation), DukeUniversity rapid autopsy program (0-3 hours postmortem), NationalDisease Research Interchange (Philadelphia, Pa.; 0-5 hours postmortem),and the International Institute for the Advancement of Medicine(Scranton, PA; within 12 hours postmortem). Except for functionalassays, vessels were snap frozen in liquid nitrogen and stored at −70°C. for later use.

[0022] Membrane Preparation and Radioligand Binding

[0023] Vessels were weighed, lumen diameter measured, pulverized underliquid nitrogen, and suspended in cold lysis buffer (5 mmol/L Tris HCland 5 mmol/L EDTA, pH 7.4) with protease inhibitors.¹⁴ After lysatepreparation, membranes were resuspended in cold binding buffer (150mmol/L NaCl, 50 mmol/L Tris·HCl, 5 mmol/L EDTA, with proteaseinhibitors, pH 7.4) as previously described;¹⁴ protein concentration wasdetermined using the bicinchoninic acid method (Pierce, Rockford, Ill.).Full saturation binding isotherms were performed in selected humanvessels (aorta, mammary artery, saphenous vein) in 250 μl binding buffer(20-60 μg vessel membrane protein) using the α₁-adrenergic antagonist[¹²⁵I]HEAT(2-[b-(hydroxy-3[¹²⁵I]iodophenyl)ethyl-aminomethyl]-tetralone;DuPont-NEN; Boston, Mass.) as previously described.¹⁴ To measure totala₁AR density in all vessels, a saturating concentration (300 pmol/L) ofthe [¹²⁵I]HEAT was used. A Kd concentration (130 pmol/L [¹²⁵I]HEAT) wasused in competition analysis with antagonists 5-MU WB4101, and BMY7378(10⁻¹² to 10⁻⁴ mol/L).

[0024] RNase Protection Assays (RPAs)

[0025] RNA isolation and human a₁AR cDNA constructs have previously beendescribed.¹⁴ RPAs were performed as previously described; controlb-actin consisted of 0.104 kb (HinP1l/Taq;) fragment in pGEM-4Z (GenBank#AB004047; nucleotide 119-222).¹⁴ [³²P]aCTP (DuPont-NEN) wasincorporated into RNA probes at the time of synthesis. After digestionwith RNase A and T1, RNA samples were separated electrophoreticallythrough a 6% polyacrylamide gel, dried, and exposed to X-Omat film(Eastman Kodak Company; Rochester, N.Y.) for 18-24 hours, andPhosphorlmager plates (Molecular Dynamics: Sunnyvale, Calif.) for 72hours. Volume integration of protected fragments was corrected forbackground using ImageQuant image analysis software (Molecular Dynamics)and counts were normalized for b-actin signal and ³²P-aCTP incorporation(CTPs: a_(1a)—97, a_(1b)—219, a_(1d)—133). Final mRNA data are scaled +1to +10, with +10 (100 arbitrary units) assigned a_(1a)AR mRNA in liver(human tissue known to contain maximal a₁AR mRNA); thus Phosphorimagercounts/10,000×1.8 defined Phosphorimager units. a_(1a)AR mRNA is highestin mesenteric artery (26 units): therefore, +3=20-29 units; +2=10-19units; +1=4-9 units; (−)=almost undetectable signal (≦3 units)Phosphorimager, negative autoradiograph; −=lack of signal on both.

[0026] Functional Assays

[0027] Since the presence of receptor protein does not always correlatewith functional response,¹⁵ a₁AR-mediated contractility in mammaryartery was tested using phenylephrine dose response curves in theabsence/presence of subtype selective/nonselective antagonists. Mammaryarteries were immersed in cold oxygenated Krebs-Ringer bicarbonatesolution (118.3 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO₄, 1.2mmol/L KHPO₄, 42.5 mmol/L CaCl₂, 25 mmol/L NaHCO₃, 16 mmoVL CaEDTA, 1.1mmol/L glucose), cleaned of loose connective tissue, cut into 4-5 mmlong rings, and suspended for isometric tension recording in organchambers. One stirrup was anchored to the chamber and the otherconnected to a strain gauge (FT-102) for measurement of isometric force(MacLab, CB Sciences; Milford, Mass.). All concentration effect curveswere performed at optimum resting tone (˜3 g in pilot studies).Contractile response to 60 mmol/L KCI was performed; this determinedvessel viability and facilitated normalization of phenylephrine responseacross vessel rings. Phenylephrine dose-response curves were generated(10⁻⁴-10⁻⁹mol/L) in ½ log order concentrations in the absence/presenceof competitive a₁AR antagonists. Contraction assays using vessel ringsfrom an individual patient were performed simultaneously in separatebaths for each antagonist; hence each vessel ring was exposed to threedose response curves. Antagonist potency was expressed as thedissociation constant (K_(B)) determined from pK_(B)=log[B]log(DR−1),where [B] is antagonist concentration and DR the dose ratio produced byantagonist. Dose response curves were analyzed using DOSE RESPONSEsoftware (MacLab, CB Sciences).

[0028] Statistical Analysis

[0029] Data were tested for normal distribution using Shapiro-Wilke testof normality. Overall α₁AR density was compared between vessels using ageneral linear multivariate model, and where significant differencesidentified between specific vascular beds, the exact p value wasdetermined using Wilke's-Lambda test; p<0.05 was considered significant.Since determination of α₁AR subtype expression involved three subtypes(a_(1a), a_(1b), a_(1d)), critical α was reduced to 0.0167 for thesestudies. Similarly, when comparing α₁AR subtype expression betweendifferent vascular beds, pairwise comparisons were made using a Wilcoxon2-sample rank sum test, and critical α set at 0.0167. Competitionbinding and functional assays were analyzed using least squaresregression analysis with Prism software (GraphPad; San Diego, Calif.).Final data were analyzed using SAS system, release v.6.12 (SAS InstituteInc., Cary, N.C.), and presented as mean±SEM to two significant figures.

[0030] Results

[0031] Characterization of Human Vessels

[0032] 500 vessels from 384 patients (male, n=257; female, n=127;64±0.82 years [range 12-92]) were used. The majority (83%) werecollected from operating room specimens, 17% from autopsy (cause ofdeath: gun shot, automobile accident, myocardial infarction, cancer).95% vessels were obtained ≦3 hours from tissue isolation or death(within 12 hour postmortem mRNA/protein stability period inrats/humans).^(16,17) Vessels were obtained only from patients withoutco-existing disease (e.g. no chronic renal failure, congestive heartfailure, diabetes, hypertension, thyroid disease), or potentiallyconfounding drugs (e.g. no estrogen supplementation, catecholamines,sympathetic stimulants, antidepressants, or aAR drugs); five years wasrequired to collect enough vessels to complete the study. Due to limitedvessel RNA/protein, n=1 vessel from a single individual wheneverpossible, but sometimes represents pooled samples from 2-6 patients withsimilar patient characteristics.

[0033] Human Vascular Total a₁AR Expression

[0034] The “fight and flight” (stress) response results inredistribution of blood from splanchnic and “non-essential” organstoward vital organs.^(2,3) In order to test the hypothesis that a₁ARdensity in splanchnic versus somatic vessels may be responsible forthese effects, Kd and Bmax were determined for ¹²⁵I-HEAT binding inselected human vessels (nonspecific binding 30-70%). Kd is 130±0.20(aorta), 130±3.1 (mammary artery), and 130±0.65 (saphenous vein) pmol/L(n=2-4 each, FIG. 1), similar to cloned human a₁ARs.⁴ Overall humanvascular a₁AR expression is 16±2.3 fmol/mg total protein; central(conduit) and small somatic arteries express significantly lower a₁ARdensity than splanchnic arteries, p<0.05, Table 1). In contrast, venousa₁AR density does not change with vessel diameter or vascular bed.

[0035] a₁AR Subtype mRNA in Human Vessels

[0036] a₁AR subtypes were next examined; due to limited tissue,molecular approaches were utilized. All three a₁AR mRNAs are present inhuman vessels (FIG. 2), with α_(1a)AR predominating overall in arteries(p<0.001); epicardial coronary arteries express ala exclusively (Table2). α_(1a)AR subtype density is significantly higher in splanchnicversus central vessels (p<0.05; FIG. 3). These findings suggest a₁ARsubtype expression varies with vessel type.

[0037] a₁AR Subtype Protein in Human Vessels

[0038] To ensure mRNA and protein expression correlate, competitionanalysis was performed. Selected vessels were chosen for availabilityand expression of only one or two α₁AR subtypes (to facilitateinterpretation of results). Since a_(1a)AR mRNA predominates, 5-MU(a_(1a)-selective antagonist) was utilized. a₁AR subtype proteinexpression in 4 representative human vessels was determined bycompetition analysis with 5-MU (a_(1a)-selective antagonist) (n=3experiments per vessel, each performed in triplicate); Table 3summarizes pki values (−logKi; measure of receptor affinity forantagonist). 5-MU binds to two sites in mammary, renal, splenicarteries, and vena cava, with the high affinity pKi site consistent withinteractions at cloned α_(1a)ARs.⁴ Although designation of the highaffinity binding site is straightforward, low affinity α₁AR siteidentification was aided by mRNA data in Table 2 and confirmed inmammary artery (and aorta) using BMY7378 (a_(1d)-selective antagonist).Only one binding site was detected in aorta, coronary artery, andhepatic artery, with pKi values consistent with α_(1d), α_(1a), andα_(1a)ARs, respectively. These data suggest mRNA and protein expressioncorrelate closely in human vessels.

[0039] Human Mammary Artery Contraction

[0040] Phenylephrine dose response curves were completed in 10 mammaryarteries (patient age 60±2.2 years [range 37-73]), vessels which containonly a_(1a) and a_(1b)ARs; isometric contraction occurs with pD₂6.0±0.093. a₁AR competitive antagonists produce a concentrationdependent shift in potency of phenyiephrine contraction without reducingmaximum response (FIG. 4). Potency in inhibiting mammary arterycontraction (pK_(B)) is 9.2±0.046 (prazosin, non-selective), 8.4±0.63(5-MU. a_(1a)-selective), and 8.6γ0.19 (spiperone, relativelya_(1b)-selective), similar to affinities for each antagonist at clonedhuman a₁ARs.⁴ BMY7378 (α_(1d)-selective) does not produce a shift indose response. These data suggest a_(1a) and a_(1b)ARs mediatecontraction in human mammary artery.

[0041] Regulation of Vascular a₁AR Subtype Expression by Age

[0042] Mammary artery α₁AR density increases significantly with age(4.4±0.78<55 years versus 9.3±1.7≧65 years, p=0.003, fmol/mg totalprotein) (Table 4). In contrast, saphenous vein α₁AR density does notchange with age. Competition analvsis with 5-MU and WB4101 revealsα_(1a)ARs are the major subtype in mammary artery in patients <55 yearsof age (FIG. 5). However, with aging, α_(1b)AR expression significantlyincreases (3-fold, p=0.0001), becoming the major subtype in patients ≧65years; α_(1a)ARs also significantly increases with age (1.5-fold,p≦0.001). α_(1d)AR expression is virtually absent in younger and olderpatients.

REFERENCES

[0043] 1. Ruffolo R R, Jr. Distribution and function of peripherala-adrenoceptors in the cardiovascular system. Pharmacol Biochem Behav.1985;22:827-833.

[0044] 2. Allen K B, Salam A A, Lumsden A B. Acute mesenteric ischemiaafter cardiopulmonary bypass. J Vasc Surg. 1992;16:391-396.

[0045] 3. Reilly P M Bulkley G B. Vasoactive mediators and splanchnicperfusion. Crit Care Med. 1993;21:S55-S68.

[0046] 4. Schwinn D A, Johnson G I, Page S O, Mosley M J, Wilson K H,Worman N P, Campbell S, Fidock M D, Furness M, Parry-Smith D, Peter B,Bailey D S. Cloning and pharmacological characterization of human alphaladrenergic receptors. J Pharmacol Exper Ther. 1995;272:134-142.

[0047] 5. Hieble J P, Bylund D B, Clarke D E, Eikenburg D C, Langer S Z,Lefkowitz R J, Minneman K P, Ruffolo R R, Jr. International union ofpharmacology. X. Recommendation for nomenclature of a₁-adrenoceptors.Pharmacol Rev. 1995;47:267-270.

[0048] 6. Price D T, Lefkowitz R J, Caron M G, Berkowitz D, Schwinn D A.Localization of mRNA for three distinct a₁-adrenergic receptor subtypesin human tissues. Mol Pharmacol. 1994;45:171-175.

[0049] 7. Price D T, Chari R S, Berkowitz D E, Myers W C, Schwinn D A.Expression of a₁-adrenergic receptor subtype mRNA in rat tissues andhuman SK-N-MC neuronal cells. Mol Pharmacol. 1994;46:221-226.

[0050] 8. Graham R M, Perez D M, Hwa J. Piascik M T. a₁-adrenergicreceptor subtypes. Molecular structure, function, and signaling. CircRes. 1996;78:737-749.

[0051] 9. Leech C J Faber J. Different a-adrenoceptor subtypes mediateconstriction of arterioles and venules. Amer J Physiol.1996;270:H710-H722.

[0052] 10. Kong J-Q, Taylor D A, Fleming W W. Functional distributionand role of alpha₁ adrenoceptor subtypes in the mesenteric vasculatureof the rat. J Pharmacol Exper Ther. 1994;268:1153-1159.

[0053] 11. Xin X, Yang N, Eckhart A D, Faber J E. a_(1D)-adrenergicreceptors and mitogen-activated protein kinase mediate increased proteinsynthesis by arterial smooth muscle. Mol Pharmacol. 1997;51:764-775.

[0054] 12. Rokosh D G, Stewart A F, Chang K C, Bailey B A, Karliner J S,Camacho S A, Long C S, Simpson P C. a₁-adrenergic receptor subtype mRNAsare differentially regulated by a₁ -adrenergic and other hypertrophicstimuli in cardiac myocytes in culture and in vivo: repression of a_(1B)and a_(1D) but induction of a_(1C) . J Biol Chem. 1996;271 :5839-5843.

[0055] 13. Hu Z W, Shi X Y, Hoffman B B. Insulin and insulin-like growthfactor I differentially induce a₁-adrenergic receptor subtype expressionin rat vascular smooth muscle cell. J Clin Invest. 1996;98:1826-1834.

[0056] 14. Malloy B J, Price D T, Price R R, Bienstock A M, Dole M K,Funk B L, Donatucci C F, Schwinn D A. a₁-adrenergic receptor subtypes inhuman detrusor. J Urol. 1998;160:937-943.

[0057] 15. Piascik M L, Guarino R D, Smith M S, Soltis E E, Saussy D L,Jr, Perez D M. The specific contribution of the novel alpha_(1D)adrenoceptor to the contraction of vascular smooth muscle. J PharmacolExper Ther. 1995;275:1583-1589.

[0058] 16. Johnson S A, Morgan D G, Finch C E. Extensive postmortemstability of RNA from rat and human brain. J Neurosci Res.1986;16:267-280.

[0059] 17. Sherwin A, Feindel W, Andermann F, Robitaiile Y, GuevremontD, Reader T. Stability of a₁-adrenoceptors in surgically excised humanbrain. Life Sci. 1986;39:953-958.

[0060] 18. Little R A Kirkman E. Cardiovascular control after injury.Edited by Cooper G J, Dudley H A F, Gann D S, Little R A. Maynard R L.Oxford, ed., 1997, pp.551-563

[0061] 19. Piascik M T, Hrometz S L, Edelmann S E, Guarino R D, Hadley RW, Brown R D. Immunocytochemical localization of the alpha-1 Badrenergic receptor and the contribution of this and other subtypes tovascular smooth muscle contraction. J Pharmacol Exper Ther.1997;283:854-868.

[0062] 20. Cavalli A, Lattion A-L, Hummier E, Nenniger M, Pedrazzini T.Aubert J-F, Michel M C, Yang M, Lembo G, Vecchione C, Mostardini M,Schmidt A, Beermann F, Cotecchia S. Decreased blood pressure response inmice deficient of the a_(1b)-adrenergic receptor. Proc Natl Acad SciUSA. 1997;94:11589-11594.

[0063] 21. Muramatsu I, Kigoshi S, Ohmura T. Subtypes of a₁-adrenoceptorinvolved in noradrenaline induced contractions of rat thoracic aorta anddog carotid artery. Jap J Pharmacol. 1991-57:535-544.

[0064] 22. Suzuki E, Tsujimoto G, Tamura K, Hashimoto K. Twopharmacologically distinct a₁-adrenoceptor subtypes in the contractionof rabbit aorta. Mol Pharmacol. 1990;38:725-736.

[0065] 23. Shibata K, Hirasawa A, Foglar R, Ogawa S. Gozoh T. Effects ofquinidine and verapamil on human cardiovascular a₁-adrenoceptors.Circulation. 1998;97:1227-1230.

[0066] 24. Diehl N L Shreeve S M. Identification of thea_(1c)-adrenoceptor in rabbit arteries and the human saphenous veinusing the polymerase chain reaction. Eur J Pharmacol. 1994;268:393-398.

[0067] 25. Hatano A, Takahashi H, Tamaki M, Komeyama T, Koizumi T,Takeda M. Pharmacological evidence of distinct a₁-adrenoceptor subtypesmediating the contraction of human prostatic urethra and peripheralartery. Br J Pharmacol. 1994;113:723-728.

[0068] 26. Testa R, Guarneri L, Taddei C, Poggesi E, Angelico P, SartaniA, Leonardi A, Gofrit O N, Meretyk S, Caine M. Functional antagonisticactivity for Rec 15/2739, a novel a₁ antagonist selective for the lowerurinary tract, on noradrenaline-induced contraction of human prostateand mesenteric artery. J Pharmacol Exper Ther. 1998;277: 1237-1246.

[0069] 27. Gurdal H, Tilakaratne N, Brown R D, Fonseca M, Friedman E,Johnson M D. The expression of alpha₁ adrenoceptor subtypes changes withage in the rat aorta. J Pharmacol Exper Ther. 1995;275:1656-1662.

[0070] 28. Xu K M, Tang F, Han C. Alterations of mRNA levels ofa₁-adrenoceptor subtypes with maturation and ageing in different ratblood vessels. Clin Exper Pharmacol Physiol. 1997;24:415-417.

[0071] 29. Shaul P W, Magness R R, Muntz K H, DeBeltz D, Buja L M.a₁-adrenergic receptors in pulmonary and systemic vascular smoothmuscle. Circ Res. 1990;67:1193-1200.

[0072] 30. Ibarra M, Terron J A, Lopez-Guerrero J J, Villalobos-MolinaR. Evidence of an age-dependent functional expression ofa_(1D)-adrenoceptor in the rat vasculature. Eur J Pharmacol.1997;322:221-224.

[0073] 31. O'Connor G T, Plume S K, Olmstead E M, Coffin L H, Morton JR, Maloney C T, Nowicki E R, Levy D G, Tryzelear J F, Hernandez. F,Adrian L, Casey K J, Bundy D, Soule D N, Marrin C A S, Nugent W C,Charlesworth D C, Clough R, Katz S, Leavirt B J, Wennberg J E.Multivariate prediction of in-hospital mortality associated withcoronary artery bypass graft surgery. Circulation. 1992;85:2110-2118.

[0074] 32. Christenson J T, Schmuziger M. Maurice J, Simonet F, VelebitV. Gastrointestinal complications after coronary artery bypass grafting.J Thorac Cardiovasc Surg. 1994:108:899-906.

[0075] 33. Barnett M G, Swartz M T, Peterson G J, Naunheim K S,Pennington D G, Vaca K J, Fiore A C, McBride L R. Peigh P, Willman VLea.Vascular complications from intraaortic balloons: risk analysis. J VascSurg. 1995;19:81-87.

[0076] 34. Buzelin J M, Fonteyne E, Kontturi M. Witjes W P J, Khan A.Comparison of tamsulosin with alfuzosin in the treatment of patientswith lower urinary tract symptoms suggestive of bladder outletobstruction (symptomatic benign prostatic hyperplasia). Br J Urol.1997;80:597-605.

[0077] All documents cited above are hereby incorporated in theirentirety by reference.

[0078] One skilled in the art will appreciate from a reading of thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

What is claimed is:
 1. A method of preventing restenosis aftermyocardial infarction and reprofusion comprising administering to apatient in need of such prevention an α_(1a)-adrenergic receptor(α_(1a)-AR) or α_(1a)/α_(1d)-AR selective antagonist in an amountsufficient to effect said prevention.
 2. A method for screening a testcompound for the ability to prevent restenosis after myocardialinfarction and reprofusion comprising assaying said compound foractivity as an α_(1a)-AR or α_(1a)/α_(1d)-AR selective antagonist.